The present invention relates to a method for the determination of free radicals through the quantification of their quenching products. More particularly, the invention relates to a method which allows the determination of free radicals in any kind of clinical chemistry analyzer, such determination being possible in any laboratory having commonly available standard instrumentation.
As is well known to those skilled in the art, the term xe2x80x9cfree radicalxe2x80x9d is meant in the literature to include a chemical species able to exist independently and containing one or more unpaired electrons in the outermost atomic and/or molecular orbital.
Of the existing free radicals, those referred to as xe2x80x9coxygen-centered free radicalsxe2x80x9d (or more simply xe2x80x9coxygen-centered radicalsxe2x80x9d) have attracted increasing interest in recent years in the medical field in view of the role they can assume for determination of tissue damage of different pathological conditions.
It should be pointed out that carbon centered radicals R. formed under aerobic conditions are quickly converted by oxygen to peroxy radicals ROO. which under reaction conditions are converted to hydroperoxides ROOH through hydrogen abstraction or, via dimerisation, to tetra-oxides ROOOOR, which in turn quickly fragment to alkoxy radicals RO. and oxygen.
Characteristic of these oxygen-centered radicals is to have an odd number of electrons in the oxygen outermost orbit, which makes them very reactive toward a great number of biologically relevant molecules.
In English language clinical medical literature, these radicals, the diamagnetic species originating from them, as well as their metabolites, are commonly called ROMs (Reactive Oxygen Metabolites). The main ROMs include the oxygen superoxide radical anion (O2xe2x88x92.), alkoxy radicals (RO.), peroxy radicals (ROO.), hydroxy radical (HO.), hydrogen peroxide (H202), and hydroperoxides (ROOH).
Recent studies have indicated several clinical conditions where the ROMs are invoked or recognized to play a major role. In particular, these include inflammatory and post-ischemia diseases of the alimentary tract, central nervous system alterations resulting from vascular or traumatic disorders, atherosclerosis and its cardiac complications, some renal disorders, rheumatoid and deformans arthritis, and alcohol-induced disorders in specific parts of the organism. Under physiological conditions, free radicals are extremely labile (short-lived) and remain elusive species that cannot be detected, let alone quantified. Thus their pathogenic role in the above diseases has been deduced only by following the effects exerted by added antioxidants which could not have a specific anti-inflammatory activity of their own.
Even Electron Spin Resonance spectroscopy, which is the technique of choice for the direct detection and characterization of free radicals, is not normally successful in biological systems. Because this technique requires extremely sophisticated and expensive instruments, it cannot be adopted by diagnostic (analytic) laboratories for daily routine determinations.
This makes extremely important the herein defined simple methodology (protocol) based on the use of the present invention for the determination of free radicals and of their derivatives in samples of different natures, and especially in biological samples where the invention would allow the monitoring of the effects of antioxidant and anti-inflammatory therapies through analysis that can be carried out in any standard laboratory.
The applicant has been able to determine on an experimental basis that N, N-diethyl-para-phenylenediamine [(C2H5)2NC6H4NH2], hereafter indicated as chromogen, when used in an appropriate concentration in a buffer of suitable pH where a biological sample has been previously dissolved, undergoes a chromogenic reaction making it possible to determine the free radicals initially present in the biological sample through the hydroperoxides resulting from their quenching.
The red color developed in the chromogenic reaction is characterized by absorption peaks at 510 nm and 550 nm, as can be noted in graph 1, and perfectly follows the Lambert and Beers law at both wavelengths, as evidenced in the enclosed graph 2.
The invention is based on the fact that, as already mentioned, the free radicals formed in a biological system are eventually converted to hydroperoxides. When the biological sample is dissolved in the acidic buffer, the hydroperoxides are converted to alkoxy and peroxy radicals by the metal ions released by the proteins. These radicals receive an electron from the added chromogen which is therefore converted to the corresponding radical cation responsible for the red color.
If the sample is not of biological origin, the method must be modified by adding a third reagent (C), in particular a transition-metal ion salt, preferably an Iron (II) salt, in order to provide the transition-metal ions necessary to bring about the degradation of the hydroperoxides. It should be emphasized that in either case the presence of EDTA or other agents known to complex transition metal ions in the examined solution must be absolutely avoided, because it would inhibit the degradation of hydroperoxides.
The ROMs determination in blood, serum, liquor, etc. by the method of this invention can be made either by reading the absorbance (at 505 or 550 nm) when the color is fully developed (End Point reading), or by determining the rate of variation of the color in a predetermined time interval (Fixed Point reading).
The great advantage of this method over those presently known is that it does not require special apparatuses and can be used with the instrumentation already present in any laboratory.
The fact that the method proposed by the Applicant and using the method according to the invention requires the use of two separated reagents A and B, where A represents the chromogen and B a suitably buffered solution, is not a limitation: the two reagents are mixed in an appropriate proportion to make a sole working reagent. When reagent C is necessary, it should be added to the final buffer solution resulting from the mixing of A and B and already containing the sample to be examined.
It is therefore a specific object of the present invention to present a method for the determination of ROMs comprising a chromogen chosen from the following group: 
where R1, R2, R3, and R4xe2x95x90CH2CH3, CH3, H, or X, with X being a halogen atom, the chromogen (A) being dissolved in water or other suitable solvent, and a buffer (B) having a pH value in the range 3 to 6.9.
Preferably, the chromogen and the buffer are mixed with a proportion between 1:20 and 1:300.
According to a first preferred embodiment of the invention, the chromogen comprises N, N-diethyl-para-phenylenediamine.
In a second preferred embodiment of the method according to the invention, the chromogen can comprise N, N-diethyl-paraphenylenediamine sulfate, [C10H16N2xc3x97H2SO4].
Still according to the invention, in a third preferred embodiment, said chromogen can comprise N, N-diethyl-para-phenylenediamine oxalate, [C10H16N2xc3x97C2H2O4].
Further according to the invention, said chromogen can comprise N, N-dimethyl-para-phenylenediamine [CH8H12N2], N, Ndimethyl-para-phenylendiamine sulfate, [C8H12N2xc3x97H2SO4], or N, Ndimethyl-para-phenylenediamine oxalate, [C8H12N2xc3x97C2H2O4].
Preferably, according to the invention, said buffer (reagent B) will have a pH value between 4 and 5.
According to a preferred embodiment of the method according to the invention, a third reagent (C) is added, in particular a transition metal ion salt, preferably an Iron (II) salt in order to provide the transition-metal ions necessary to bring about the degradation of the hydroperoxides.
Preferably, said reagent C will be an aqueous solution of Iron (II) sulfate.
Furthermore, said reagent C shall be an aqueous solution of cuprous (I) salt, preferably cuprous sulfate.